Transplantation and survival of mouse inner ear progenitor/stem cells in the organ of Corti after cochleostomy of hearing-impaired guinea pigs: preliminary results.

In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350 g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

Transplantation and survival of mouse inner ear progenitor/stem cells in the organ of Corti after cochleostomy of hearing-impaired guinea pigs: preliminary results

In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.

Vibratome sectioning for enhanced preservation of the cytoarchitecture of the mammalian organ of Corti.

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in (1,2)).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques. Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see (3-6) for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice (7). This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.

Effects of Piezosurgery on the cochlear outer hair cells.

CONCLUSIONS: The absence of audiologic side effects highlights the reduced trauma of the piezoelectric cut, demonstrates the superiority of the Piezosurgery device in terms of safety and protection of anatomical structures and confirms its applicability in all the otologic techniques tested. OBJECTIVES: The aim of the present study was to estimate the effect of Piezosurgery on the cochlea and in particular on the cochlear outer hair cells. PATIENTS AND METHODS: We selected 60 patients with a history of otologic surgery with Piezosurgery. Before and 6 months after surgery, all the patients underwent the following instrumental examinations: pure-tone audiometry, tympanometry, transient evoked otoacoustic emissions (TEOAEs), distortion product otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR). RESULTS: Piezosurgery showed its safety on the inner ear and in particular on the cochlear outer hair cells: for each instrumental examination (pure-tone audiometry, tympanometry, TEOAE, DPOAEs and ABR), no patients presented postoperative worsening.

Preservation of basal inner ear structures in cochlear implantation.

The aim of this report was to examine basal trauma in implanted human temporal bones and discuss modified approaches to the basal cochlear turn to avoid destruction of basal cochlear structures. Thirty-three human temporal bones were implanted with four different cochlear implant electrode arrays manufactured by MED-EL using either a caudal approach cochleostomy or round window membrane insertions. All specimens were processed with a special histological technique that allows sectioning of undecalcified bone with the electrode in situ. All bones were evaluated histologically in terms of basal cochlear trauma. Two pathomechanisms of basal trauma could be distinguished and were evaluated separately, buckling of the basal end of the array and trauma by drilling. Using the caudal approach cochleostomy, the total percentage of destructive basal trauma was 48% compared to less than 15% when performing round window membrane insertions. Although it is still unclear whether basal cochlear trauma influences apical cochlear function or not, adapted surgical procedures and no forceful insertion maneuvers should be used when performing cochlear implantations with hearing preservation.

Stapedotomy: the JB Causse technique.

The Jean-Bernard Causse technique of stapedotomy, derived from Jean René Causse "Teflon-interposition" technique, offers an optimum reconstitution of the function of the annular ligament of the stapes footplate. The quantity and quality of hearing depend significantly, among other things, on the function of the stapes footplate annular ligament. Its impedance keeps the perilymph motion within a physiological acoustic amplitude quantum level unless the movements are so excessive as in barotrauma and acoustic trauma which would have overworked even the annular ligament of a normal footplate. This surgical technique permits snorkling, scuba-diving, airplane landing, eustachian tube dysfunction after surgery as in a normal individual who did not undergo otosclerosis surgery. A comparative study on the postoperative hearing based on 3,000 out of 14,000 cases done by J C Bausse technique, 127 cases with small fenestra technique and 267 cases of complete platinectomy showed too large stapedotomy gives poor high tones and too small stapedotomy poor low tones. Five year follow-up on the hearing loss indicated that total platinoctomy has 1.7 times more alteration of 4 kHz frequency in comparison to the technique reconstituting the annular ligament. Yearly audiogram to check the evolution of cochlear otospongiosis may lead the surgeon to prescribe sodium fluoride if the mid-frequencies are deteriorating and vascular drugs if the high pitch frequencies are deteriorating.

Reversible damage to the nerve fibres in the organ of Corti after surgical opening of the cochlea in the rat.

After minimal opening of the cochlear bony wall, the efferent and the outer spiral fibres showed no changes; inner radial fibres (afferents to inner hair cells) were highly sensitive to this mild trauma, appearing swollen and empty of cytoplasmic content. Available data suggest that this may be due to alterations in the cochlear micromechanical environment, related to the surgical manipulation. The swellings were reversible, although the normal structure had not completely recovered until one month after the manipulation.

Electrolytic lesioning in the cochlea.

A method has been developed whereby relatively discrete protions of the organ of Corti can be selectively destroyed with the use of a high-frequency current. The procedure involves passing th current (1 to 3 ma at an approximate frequency of 5 megahertz) between a differential pair of recording electrodes located in the scalae vestibuli and tympani of a particular cochlear turn. Damage patterns progressing from partial to complete outer hair cell lesioning and eventually to the inner hair cells accompanied by progressive decreases in cochlear microphonic sensitivity can be effected.